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The aim of this study was to adapt the Comet assay in spermatozoa of the marine prawn Palaemon serratus to use it as a marker of sperm quality. Indeed, due to the characteristics of their spermatozoa, the measurement of DNA integrity is one of the few markers which can be transferred to crustaceans to assess the quality of their semen. In the first step, the methods of collecting and maintaining spermatozoa were optimized. Cell survival was estimated during kinetics of preservation (i.e. 1, 2, 4 and 8 h) in various suspension media to define artificial seawater (ASW) as optimal. Several methods in the releasing of spermatozoa from the spermatophore of prawns were estimated with regard to their incidence both on the efficiency of extraction and the survival of cells. Pipetting up and down turned out to be the most successful and the least invasive technique. Secondly, the transfer of Comet assay was optimized by studying various times in both cell lysis (i.e. 1, 6, 18 h) and DNA denaturation (i.e. 15, 30 and 45 min), after in vitro exposure of spermatozoa to an H2O2 gradient as model genotoxicant. Results revealed that a minimum of 1 h in cell lysis and 15 min of DNA denaturation were sufficient to obtain valuable results, linked with a low compaction of DNA in spermatozoa of Palaemon sp. Finally, the sensitivity of P. serratus spermatozoa was assessed after in vitro exposures to model genotoxicants displaying various modes of interaction with DNA (i.e. UV-C, 13.3-79.5 J m; HO, 5-10 μM and MMS, 0.5-5 mM) and some environmental contaminants known or suspected to be genotoxic (i.e. cadmium and diuron, 0.015-1.5 μg L; carbamazepine, 0.1-10 μg L) for invertebrates. The low variability of the baseline level of DNA strand breaks recorded in controls highlighted the robustness of the method. P. serratus spermatozoa displayed significant DNA damage from the lowest doses tested for all model genotoxicants, but conversely, no genotoxic effect of tested environmental contaminants was observed. These results, which are discussed according to the protocol tested in the present study and the comparison with literature data, could suggest a difference in the response or sensitivity of spermatozoa to environmental genotoxicity between invertebrate species, and therefore the interest of Palaemonidae prawns in ecogenotoxicology. In conclusion, the present study underlines the potential of the Comet assay as a marker to assess the contamination impact on the sperm quality in Palaemonidae prawns in view to a potential application for in situ biomonitoring surveys.

In a previous study, the Comet assay was optimized for Palaemon serratus prawns in order to propose a biomarker for sperm quality in this species. However, better knowledge of its basal level and its natural variability, related to intrinsic biotic and environmental abiotic factors, is required before any relevant use of this biomarker in the field. To fulfill this goal, the present study proceeded in three steps: (i) the temporal variability of DNA integrity was followed monthly in a reference population over a 2-year period, (ii) the correlation between the main intrinsic biotic (i.e. size, weight and molting stage) and abiotic factors (i.e. water temperature) were recorded in the field, and the basal DNA integrity was assessed in order to scrutinize any confounding influence of factors unrelated to toxic response, (iii) the baseline level was used to discriminate biomarker response among different stations displaying contrasting contamination levels. The results of the two-year monitoring in the reference population revealed no correlation between the levels of spermatozoa DNA damage and temperature, body size, weight or molting stage. Only a slight variability between monthly samplings was detected. On the basis of these field-collected data, we defined a reference distribution (i.e. 52.6 ± 5.6 A.U) with a threshold value (i.e. 61.7 A.U). Finally, this threshold value proved its relevance to discriminate among stations with contrasting pollution levels around the Seine Bay. Indeed, the results suggest significant DNA damage in populations nearest the Seine estuary, a major source of contaminants in the Bay, and a lower effect in populations further away from the estuary. The overall conclusion was that the Comet assay on P. serratus spermatozoa could be a useful tool for the monitoring of the toxicological print within sperm and main globally the contamination exposure of crustaceans in marine waters.

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The interpretation of biomarkers in natura should be based on a referential of expected values in uncontaminated conditions. Nevertheless, to build a reference data set of biomarker responses in estuarine areas, which receive chronic pollution loads due to their transition position between continent and sea, is impossible. In this context, the aim of the present work was to propose the use of laboratory recovery period to define a baseline for the measurement of sperm DNA damage by Comet assay in the estuarine prawn Palaemon longirostris. For that, sperm DNA integrity was observed after both a passive (i.e. 20 days in a clean environment) and an active (i.e. forced renewal of spermatophores) recovery of wild P. longirostris specimens from the Seine estuary, in laboratory conditions. Then, the levels of sperm DNA damage recorded within the P. longirostris population of the Seine estuary, during six campaigns of sampling from April 2015 to October 2017, have been interpreted according to the defined threshold values. The results showed a persistence in the level of DNA damage after 20-day in clean environment with the passive recovery. This strategy was inconclusive to reach a baseline level but it revealed the lack of DNA repair mechanisms. For the active recovery, a decrease of 54 % of the level of DNA damage has been observed after the first renewal of spermatophores and this level stabilized after the second renewal. On the basis of this second strategy, we defined a mean basal value of sperm DNA damage of 54.9 A.U. and a maximum threshold of 69.7 A.U. (i.e. 95 %-CI). The analysis of the results using the reference value highlighted significant abnormal sperm DNA damage within the native population of P. longirostris from the Seine estuary on all stations during the six-sampling campaigns.

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